Immunoproteasome modulates NLRP3 inflammasome‐mediated neuroinflammation under cerebral ischaemia and reperfusion conditions

Abstract Compelling evidence showed that both nucleotide‐binding oligomerization domain‐like receptor family, pyrin domain‐containing protein 3 (NLRP3) inflammasomes and the immunoproteasome participate in neuroinflammatory responses in cerebral ischaemia injury. Moreover, inhibition of either NLRP3 inflammasomes or the immunoproteasome attenuates both neuroinflammation and neurological deterioration during ischaemic stroke. However, the underlying mechanism between the immunoproteasome and NLRP3 inflammasomes under ischaemic stroke conditions remains to be established. In this study, using both in vitro and in vivo ischaemic models, we demonstrated that the immunoproteasome inhibition reduced the expressions of NLRP3 inflammasome‐associated proteins, including NLRP3, apoptosis‐associated speck‐like protein (ASC), caspase‐1 and mature cytokines (interleukin [IL]‐1β and IL‐18). It also downregulated the levels of nuclear factor (NF)‐κB and pyroptotic‐ and apoptotic‐related proteins, and improved cell viability. In addition, inhibition of NF‐κB by the small molecule inhibitor Bay‐11‐7082 led to lower levels of NLRP3 inflammasomes and cleaved caspase‐1 proteins in BV2 cells after oxygen‐glucose deprivation and reoxygenation. Together, these findings suggest that the immunoproteasome may be responsible for inducing the expression and activation of NLRP3 inflammasomes via the NF‐κB pathway. Therapeutic interventions that target activation of the immunoproteasome/NF‐κB/NLRP3 inflammasome pathway may provide novel prospects for the future treatment of ischaemic stroke.


| INTRODUC TI ON
Stroke is the second leading cause of death worldwide and a major cause of permanent disability. Inflammation and immune responses have emerged as important factors in both the onset and progression of stroke and can shape its clinical presentation and outcome. 1 Recent research has revealed a new inflammatory mechanism that contributes to post-stroke brain damage, which is mediated by nucleotide-binding oligomerization domain-like receptor family, inflammasomes can improve the remodelling and integrity of the neurovascular unit. [5][6][7] To date, the cellular localization of NLRP3 inflammasomes in the brain remains controversial. 3 In addition, although there is evidence that nuclear factor (NF)-kB and mitogenactivated protein kinase (MAPK) signalling promotes neuronal NLRP inflammasome activation following ischaemic stroke, 8 the precise mechanisms that contribute to post-stroke NLRP3 inflammasomes activation remain unknown.
The ubiquitin-proteasome system has a central role in the selective degradation of intracellular proteins that control inflammatory processes, cell cycle regulation and gene expression. Large amounts of data suggest that the ubiquitin-proteasome system contributes to cerebral ischaemic injury, and that proteasome inhibition is a potential treatment for stroke. The proteasome complex consists of three different catalytically active subunits-β1, β2 and β5 that are constitutively expressed. Elevated levels of interferons or oxidative stress induce the expression and incorporation of the catalytically active subunits β1i/ low molecular mass polypeptide (LMP)2, β2i/LMP5 and β5i/LMP7 into newly formed immunoproteasome complexes. The catalytic β1i, β2i and β5i subunits display caspase-, trypsin-and chymotrypsin-like proteolytic activities, respectively. 9 The immunoproteasome can have either a beneficial or a detrimental role depending on the experimental context. We have previously observed that the immunoproteasome participates in inflammatory mechanisms in ischaemic stroke, while inhibition of the immunoproteasome subunit LMP2 leads to lower NF-κB expression in rat models of ischaemic stroke. 10,11 Notably, a recent study reported that the toll-like receptor 4 (TLR4)/NF-κB/ NLRP3 pathway plays an important role in sepsis-induced myocardial dysfunction. 12 We therefore hypothesized that immunoproteasome/ NF-κB/NLRP3 signalling might be involved in the inflammatory mechanisms of ischaemic stroke. In the present study, we aimed to investigate the expression of NLRP3 in the ischaemic brain and to confirm whether the immunoproteasome regulates the NLRP3 inflammasome pathway under conditions of cerebral ischaemia.

| MCAO/R model and lentiviral construction preparation and injection
Rats were anaesthetized and subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) as described previously, with minor modifications, 10,13 In brief, a midline neck incision was made, and the right common carotid artery, external carotid artery and internal carotid artery were isolated. The external carotid artery was tied.
A 4-0 monofilament nylon suture (Beijing Sunbio Biotech Co. Ltd.) with a rounded tip was aseptically inserted from the right common carotid artery to the internal carotid artery through the stump of the external carotid artery and gently advanced to occlude the middle cerebral artery. Recirculation/reperfusion of the cerebral blood flow was allowed by gently removing the monofilament after 1 h of ischaemia, followed by 72 h of reperfusion. In sham-operated animals, all procedures except occlusion of the MCA were performed.
According to our previous study, 10 the best-performing LMP2-shRNA sequence was CCTGGTCACCATTACAGCT, and the negative control scrambled shRNA sequence was CCATCATGGCTGTGGAATT (Genechem). A total volume of 10 μl of lentivirus suspension was delivered into the ipsilateral hemispheric region 3 days before MCAO using a 15 μl syringe at the following coordinates (10 μl/per site): bregma backward 1 mm, 1.5 mm lateral, 4 mm dorsoventral. 10

| Tissue preparation and TTC staining
At 72 h after reperfusion, five rats from each group were sacrificed after anaesthesia and then transcardially perfused with 0.9% sodium chloride at 4°C followed by 4% paraformaldehyde in 0.01 M phosphate-buffered saline (PBS, pH 7.4). Brains were then removed, kept in the same fixative for 48 h at 4°C and cryoprotected in serial PBS isopropanol sucrose solutions (20% and 30%) at 4°C until brains sank. Coronal sections (10 μm) were cut on a cryostat (CM1900; Leica) and used for next experiments.
The slices were stained in 2% 2, 3, 5-triphenyltetrazolium chloride (TTC) (Sigma-Aldrich Corp) for 20 min at 37°C in the dark. Then, the infarcted brain tissue appeared white, whereas the noninfarcted region appeared red. The sections were fixed with 4% paraformaldehyde and then digitized.

| Assessment of cell viability by MTT
BV2 cells viability in response to OGD/R and different treatments was assessed by3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assays. Briefly, cells were seeded in 96-well plates and were grown to about 80% confluence. 10 μl of the MTT reagent (0.5 mg/ml) was added into each well for 4 h to allow formation of purple formazan crystals. Absorbance (A) value was measured at an absorbance wavelength of 570 nm in a microplate reader within 30 min.

| Immunofluorescence staining
Brain slides and cultured cells were detected with immunofluorescence staining as described previously. 11 The following primary antibodies were used rabbit anti-NLRP3, rabbit anti-LMP2 and rat  Subsequently, TUNEL reaction mixture was then applied for 60 min at 37°C. Fluorescence signal was detected using a fluorescence microscope at excitation/emission wavelengths of 492/520 nm (FITC, green).

| Western blot analyses
Both ischaemic brain tissues and cells lysates were collected as previously described, and total protein (20-40 μg) was separated by 4-

| Measurement of 20S proteasome activity and LDH
Proteasome activity assays were performed as previously reported, 10 using a 20S Proteasome Activity Assay kit (APT280; Millipore) ac-

| Statistical analysis
The data were expressed as means ± standard deviation (SD).
Differences between groups were assessed by one-way ANOVA analysis with a Tukey's post hoc test. A value of p < 0.05 was considered statistically significant. IBM SPSS statistical software was used for statistical analysis (version 19.0; SPSS, Inc.).

| Expression and cellular localization of NLRP3 inflammasomes in the brains of MCAO rats
Because the expression and distribution of NLRP3 inflammasomes in the brain remain controversial, we first investigated NLRP3 inflammasome expression in the brains of rats after 1 h of MCAO and up to 72 h of reperfusion. After TTC staining, the infarction brain tissue appeared white, whereas the noninfarcted regions appeared red ( Figure 1A). Using immunofluorescence, NLRP3 staining was barely detectable in the cortex and striatum of sham-operated animals and in the contralateral hemisphere of ischaemic rats. In contrast, NLRP3 positivity was observed in the area surrounding the infarct core in the ipsilateral ischaemic hemisphere ( Figure 1B,C). Co-staining with 4′,6-diamidino-2-phenylindole (DAPI) revealed that NLRP3 was mostly present in the cytoplasm ( Figure 1D). To investigate the cell type(s) expressing NLRP3 inflammasomes, brain sections were then double-labelled using antibodies against cell-specific antigens. The majority of NLRP3-positive staining occurred in OX42-positive microglia/macrophage cells, although a few GFAP-positive astrocytes and NeuN-positive neurons also had NLRP3 staining ( Figure 1E,F).
Western blotting confirmed that NLRP3 inflammasome protein level was significantly elevated after cerebral ischaemia/reperfusion. Quantification of the protein confirmed an approximate fourfold increased in NLRP3 inflammasome protein level in the MCAO group compared with the sham group (p < 0.001) ( Figure 1G). Together, our findings indicate that ischaemia elevates NLRP3 inflammasome expression in brain cells, and especially in microglia.

| Inhibiting the immunoproteasome subunit LMP2 downregulates the levels of inflammasome components, proinflammatory cytokines, and apoptotic-and pyroptotic-related proteins
In the present study, we confirmed that inhibiting the immunoproteasome subunit LMP2 using shRNA led to downregulated levels of both NLRP3 inflammasome components and proinflammatory cytokines. Western blotting demonstrated that the protein levels of NLRP3, cleaved caspase-1p10, IL-1β, IL-18, NF-κB p-p65, cleaved caspase-3 p17, total GSDMD, and cleaved GSDMD-N were significantly lower in the LMP2-shRNA group than in the control-shRNA group (p < 0.001) ( Figure 3A,B).

| Effects of OGD/R on the morphology and viability of BV2 microglial cells
Oxygen-glucose deprivation/reoxygenation-treated BV2 microglial cells were enlarged and had many microspikes covering the surface of the cell body. They also displayed more intense immunoreactivity to the CD11b antigen compared with resting microglia, which had small, amoeboid shapes (provided as supplementary data Figure   S1A,B). Western blotting showed that CD11b protein expression increased following OGD/R (p < 0.001) ( Figure 4C, provided as supplementary data). Next, we investigated the effects of different durations of OGD on the viability of BV2 cells using MTT assay. After 1, 3, 6 and 9 h of OGD followed by 24 h of reoxygenation, cell viability decreased to approximately 87.8%, 67.3%, 44.6% and 32.6%, respectively (p < 0.001) (provided as supplementary data Figure   S1D). We therefore selected OGD for 3 h as the optimal duration of OGD for the next investigation.
We next revealed that the LDH content was markedly increased after OGD/R, from 623.93 ± 52.74 U/L in BV2 cells under normal culture conditions to 1550.97 ± 90.83 U/L in cells under OGD/R conditions (p < 0.001) ( Figure 4E, provided as supplementary data).

| LMP2-siRNA transfection reverses enhanced the immunoproteasome LMP2 expression and proteasome activity in BV2 cells following OGD/R
Compared with the normal culture group, immunoreactivity of the immunoproteasome subunit LMP2 was significantly enhanced in BV2 cells following OGD/R ( Figure 4A). LMP2 was unevenly distributed throughout cells, with predominant localization in the cytosolic site of the nuclear envelope/endoplasmic reticulum membrane, as observed using co-staining with DAPI ( Figure 4A). Western blotting indicated that LMP2 protein level was significantly upregulated in the OGD/R group compared with the normal culture group

| OGD/R induces NLRP3 inflammasome expression, but this effect is attenuated by LMP2-siRNA transfection
Immunofluorescent revealed that OGD/R-induced NLRP3 inflammasome expression with a predominant localization in the cytoplasm ( Figure 5A). Notably, most NLRP3-positive cells co-localized with LMP2-positive cells ( Figure 5B). Furthermore, Western blotting showed that NLRP3 protein levels were significantly higher in the OGD/R group compared with the normal culture group (p < 0.001) ( Figure 5C). However, compared with the control siRNA group, siRNA knockdown of LMP2 significantly downregulated NLRP3 protein levels in BV2 cells following OGD/R (p < 0.001) ( Figure 5D). These results indicate that inhibiting the immunoproteasome LMP2 significantly reduces NLRP3 expression under OGD/R conditions.

| Effects of immunoproteasome inhibition on the viability of BV2 cells following OGD/R
The MTT assay demonstrated that the use of LMP2-siRNA to inhibit the immunoproteasome subunit LMP2 led to better BV2 cell survival under OGD/R. The MTT absorbance value was significantly higher in the LMP2-siRNA group compared with the control siRNA group

| Inhibition of NF-κB attenuates NLRP3 inflammasome expression following OGD/R
Next, we investigated whether inhibiting the NF-κB pathway in vitro using the pharmacological inhibitor Bay-11-7082 leads to the attenuation of NLRP3 inflammasome expression, as was observed in BV2 microglial cells following OGD/R. Treatment with Bay-11-7082 reduced the protein levels of phosphorylated NF-κB p65, NLRP3 and caspase-1 compared with the vehicle control treatment (p < 0.001) (provided as supplementary data Figure S2A-D). In addition, pretreatment with Bay-11-7082 combined with LMP2-siRNA significantly downregulated the levels of phosphorylated NF-κB p65, NLRP3 and caspase-1 proteins in BV2 cells exposed to OGD/R (p < 0.001) (provided as supplementary data Figure S2E,H).

| DISCUSS ION
In the present study, ischaemia-induced NLRP3 inflammasome expression in brain cells, especially microglia, under both in vitro and in vivo ischaemic models. Furthermore, inhibition of the immunoproteasome subunit LMP2 significantly reduced the expression of NLRP3 inflammasome-associated proteins, including NLRP3, ASC, caspase-1, inflammation cytokines (IL-1β and IL-18), NF-κB and pyroptotic-and apoptosis-related proteins, and improved cell viability.
In addition, the inhibition of NF-κB with the pharmacological inhibitor Bay-11-7082 contributed to the attenuation of NLRP3 inflammasome and caspase-1 protein expression in BV2 microglial cells after F I G U R E 3 Inhibition of immunoproteasome LMP2 reduces the levels of inflammasome components, proinflammatory cytokines, apoptotic-and pyroptotic-related proteins. (A,B) Western blotting indicated that NLRP3, cleave caspase-1p10, NF-κB p-p65, IL-1β, IL-18, cleaved caspase-3 p17, full-length and cleaved GSDMD-N protein levels in each group. # p < 0.001, compared with the sham group; * p < 0.001, compared with the control-shRNA group. Sham: sham-operated group; Cont-shRNA: control-shRNA group. Data were presented as mean ± standard deviation (n = 5/group) OGD/R. These data suggest that the immunoproteasome modulates the expression and activation of NLRP3 inflammasome proteins possibly through the NF-κB signalling pathway.
The inflammasome is a vital part of the innate immune response to stroke. NLRP3 is the most intensively studied inflammasome and has been investigated in the cerebral ischaemia-or haemorrhageinduced inflammatory response. 14,15 To date, the expression and distribution of NLRP3 inflammasomes in the brain remain controversial. Both our and other studies have indicated that NLRP3 is mainly expressed in microglia and is hardly detected in neurons and astrocytes. This indicates that microglia might be a major producer of NLRP3 inflammasomes. 16 In contrast, other studies have reported NLRP3 labelling in neurons. 17 F I G U R E 4 OGD/R significantly enhances the expression of immunoproteasome LMP2 and increases proteasome activities in BV2 cells, but was attenuated by LMP2-siRNA transfection. (A) Immunofluorescent analysis showed the expression of LMP2 under OGD/R and normal culture conditions. (B,C) Western blotting indicated that LMP2 protein levels were significantly upregulated in the OGD/R group compared with the normal culture group. * p < 0.001, compared with the normal culture group. (D,E) Compared with control siRNA group, siRNA knockdown of LMP2 significantly reduced the levels of LMP2 protein in BV2 cells exposed to OGD/R. # p < 0.001, compared with the normal culture group; * p < 0.001, compared with control siRNA group. (F) The caspase-like and chymotrypsin-like proteolytic activities in BV2 cells under OGD/R and normal culture conditions. # p < 0.001, compared with the normal culture group; * p < 0.001, compared with control siRNA group. Scale bars = 50 μm. Results were expressed as mean ± standard deviation from six independent experiments. Cont siRNA, control siRNA; OGD/R, Oxygen-glucose deprivation and reoxygenation F I G U R E 5 OGD/R induces expression of inflammasome NLRP3, but was attenuated by LMP2-siRNA transfection. (A) Immunofluorescent showed OGD/R induces expression of NLRP3 inflammasome and with a predominant localization in the cytoplasm. (B) Mostly NLRP3positive cells co-localized with LMP2-positive cells. (C) Western blotting indicated that NLRP3 protein levels in the normal culture group and OGD/R group. *p < 0.001, compared with the normal culture group. (D) Western blotting showed that NLRP3 protein levels in the normal culture, LMP2-siRNA and control siRNA group. # p < 0.001, compared with the normal culture group; * p < 0.001, compared with control siRNA group. Scale bars = 50 μm. Results were expressed as mean ± standard deviation from six independent experiments. Cont siRNA, control siRNA; OGD/R, Oxygen-glucose deprivation and reoxygenation The NLRP3 inflammasome may have a key role in detecting cellular damage and mediating inflammatory responses after stroke. 2,19 Activation of the inflammasome is usually necessary; the innate immune response to an insult efficiently protects against disease and death. However, excessive activation of the NLRP3 inflammasome can elicit a cascade of inflammatory responses, eventually leading to cell death (pyroptosis). Conversely, knockout of the NLRP3 gene or pharmacological inhibition of the NLRP3 inflammasome using MCC950 or intravenous immunoglobulin G can attenuate the expression of NLRP3 inflammasome proteins and can also downregulate IL-1β, IL-18 and proapoptotic protein cleaved caspase-3, thus largely avoiding neuronal deterioration and preserving blood-brain barrier permeability and cerebral function in ischaemic stroke models. [5][6][7] In the current study, we provided new evidence to support the idea that NLRP3 inflammasomes were upregulated in response to elevated levels of cleaved caspase-1 and caspase-3, ASC, GSDMD, IL-1β and IL-18 under in vitro and in vivo ischaemic conditions. Moreover, inhibition of the immunoproteasome subunit LMP2 led to significantly reduced levels of these proteins and improved cell viability following focal cerebral ischaemia or OGD/R. So far, the exact molecular mechanisms underlying the protective effects of NLRP3 inflammasome inhibition against cerebral ischaemia/reperfusion injury remain largely unclear. A range of complex factors and mechanisms involve in the assembly and activation of the NLRP3 inflammasome, including the NF-κB and MAPK signalling pathways, 8,20 In the present study, we also explored that the pharmacological inhibition of NF-κB decreased the NLRP3 inflammasome levels. These findings suggest that NLRP3 inflammasome expression F I G U R E 6 OGD/R induces the expression of caspase-1, ASC, NF-κB, IL-1β, IL-18, caspase-3 and GSDMD in BV2 cells, but was attenuated by genetic knockdown of LMP2. (A,B) Western blotting showed that caspase-1 p10, ASC, NF-κB p-p65, IL-1β, IL-18, cleaved caspase-3 p17, full-length GSDMD and cleaved GSDMD-N protein levels in BV2 in the normal culture, LMP2-siRNA and control siRNA group, respectively. # p < 0.001, compared with the normal culture group; * p < 0.001, compared with control siRNA group. Results were expressed as mean ± standard deviation from six independent experiments. Cont siRNA, control siRNA; OGD/R, Oxygen-glucose deprivation and reoxygenation may lie downstream of the NF-κB signalling pathway. That is, the NF-κB signalling pathway may regulate the production or activation of NLRP3 inflammasomes. Interestingly, in this study, although we found that immunoproteasome inhibition via LMP2-shRNA to some extent decreased the expression of NLRP3; results were more convincing when LMP2-shRNA and NF-κB inhibitor combined. So it seems only immunoproteasome inhibition may not be enough to suppress neuroinflammation via the NLRP3 inflammasome pathway. In addition, some studies have provided strong experimental evidence supporting a role for immunoproteasome in modulating NF-κB signalling under conditions of ischaemic stroke or inflammation response, 10,21 Immunoproteasome-mediated proteolysis has emerged as an important molecular mechanism in the regulation of wide-ranging functions, both immune (such as antigen presentation) and non-immune (including antioxidative stress), 9,22 Thus, we revealed a mechanism by which the immunoproteasome regulate the expression of NLRP3 inflammasomes by facilitating the NF-κB signalling pathway activation.
The present study has several limitations. Although we previously observed that inhibition of immunoproteasome LMP2 by shRNA reduced infarction volumes and improved the neurologic recovery in rats after 14 days ischaemia/reperfusion, 11 the neurological deficit results of these genetic and pharmacological manipulations after stroke did not provide in this study. Theoretically, neurobehavioral changes can more directly and effectively reflect the effects of these interventions. In addition, the immunoproteasome has three specific catalytic subunits LMP2, LMP5 and LMP7 with apparently diverse functions. Previous studies have reported that LMP7 expresses in microglia and plays an important role of inflammatory in some disease models. 23,24 Undoubtedly, further studies are required to fully understand the diverse roles and mechanisms by which the immunoproteasome inhibition against cerebral ischaemic injury.
In summary, the present study provides evidence that ischaemia induces the expression of NLRP3 inflammasomes, especially in microglia, and contributes to the inflammatory response and cell death. The immunoproteasome may regulate the expression of NLRP3 inflammasomes via the NF-κB signalling pathway. Together, these findings suggest that therapeutic interventions that target the immunoproteasome/NF-κB/NLRP3 inflammasome pathway may provide new opportunities for the future treatment of ischaemic stroke.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
Xingyong Chen: Conceptualization (equal); data curation (equal); formal analysis (equal); funding acquisition (equal); resources (equal); supervision (equal); validation (equal); writing -original F I G U R E 7 Inhibition of immunoproteasome LMP2 improves the viability of BV2 under OGD/R. (A) MTT assay showed cell survival under different conditions. # p < 0.001, compared with normal culture group; * p < 0.001, compared with control siRNA group. (B,C) OGD/R induced more PI-positive cells compared with normal culture group. Compared with the control siRNA group, the numbers of PI-positive cells significantly decreased in LMP2-siRNA group. (D) The levels of LDH were measured in the normal culture, LMP2-siRNA and control siRNA group. # p < 0.001, compared with normal culture group; * p < 0.001, compared with control siRNA group. Scale bars = 50 μm. Results were expressed as mean ± standard deviation from six independent experiments. Cont siRNA, control siRNA; OGD/R, Oxygen-glucose deprivation and reoxygenation draft (equal); writing -review and editing (equal). Yinzhou Wang: Conceptualization (equal); funding acquisition (equal); resources (equal). Nannan Yao: Investigation (equal); methodology (equal); resources (equal). Zejing Lin: Investigation (equal); methodology (equal); resources (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.